Prashant R. Tembhare, MD Flow Cytometry Unit Laboratory of Pathology NCI/NIH, USA

1. Flow Cytometric TCR V Beta Analysis for Determination of T cell Clonality and Its Clinical Application Prashant R. Tembhare, MD Flow Cytometry Unit Laboratory of Pathology NCI/NIH, USA

2. Flow cytometric diagnosis of T-CLPD • Altered expression of pan T cell markers like CD7, CD5, CD3, CD2 • Subset restriction CD4+ or CD8+ • Increased expression of few markers like CD25, • Aberrant Expression of antigens like CD10, CD30, CD103 Helpful in diseases with high tumor cell count

3. Problems in routine FCM • Altered expression of pan T-cell markers – Viral infections like IM, CMV, others • Subset restriction CD4+ or CD8+ may be seen in Viral infections like IM, HIV, Autoimmune diseases • Increased expression of few markers like CD25 - Increased T regulatory cells • Aberrant Expression of antigens like CD10, CD30 – limited to few NHL only - CD30+ positive T cells can be activated T cells

4. Need of T cell Clonality • Confirmation & isolation of clonal cell proliferation • Follow up of treatment – with low cell count / partially treated • Minimal Residual Disease • Low cellularity specimens like CSF, FNA deep LNs/organs staging • PB - Sezary cells • Morphology and IHC is not typical and helpful • LGLL T cell clonality usually established by molecular techniques

5. Limitations of Molecular Techniques Mainly qualitative Time Consuming Needs Specific set up Amplification of background T cells may interfere Clonal peak can not define Cell type and does not help in classification of CLPD Highly Sensitive but less specific for malignancy

6. T cell Receptor (TCR) complex T cell membrane T Cell Receptor complex Science. 1996. 274: 209-219.

7. αβ polypeptides formation Thymus SCIENCE. 272, 21 ;1996: 1755-62.

8. TCR Vβ repertoire The a TCR – chromosome 7. 65 Vβ genes – 46 are functional Vβ gene segments -26 subfamilies by cDNA -75% sequence homology at the DNA level. Palindromic & random additions and deletions Unique V-D-J = 91 – Vβ segments 24 Vβ segments usage – 70% T cells Each individual TCR-Vβ segment is expressed in only a small percentage of T cells. SCIENCE. 272, 21 ;1996: 1755-62. Immunogenetics. 1995;42(6):451-3

9. Clonal T-cell expansions are expected to express a single V domain FCA demonstration of a V restricted population is evidence of a T cell clone There are V -specific antibodies now that recognize 70% of all individual V domains – - TCR V beta Repertoire V restricted T cell populations can be quantitatively monitored during and after therapy Expanded T Cell Population - VβAnalysis

10. Anti-TCR VβAb-fluorochrome for each TCR-Vβ segment - identify a Vβ specific T cell subset Vb 7.1 FITC+PE Vb 5.3 PE PE FITC+PE FITC Vb 3 FITC

11. The IOTest Beta Mark TCR-Vβ Repertoire kit – Beckman Coulter 24 Vβ-specific antibodies - recognize 70% of all individual Vβ domains 8 tube panel ( 8 X 3 = 24) 3 antibodies in 2 colors (FITC, PE and FITC+PE) TCR Vβ Repertoire kit

12. TCR Vβ Analysis – selection of T cells

13. VβRepertoire usage in T cells from reactive lymphoid tissues % Vb usage in lymphocyte subsets Mean + 2 SD TCR Vβ Family

14. Human Immunology. 64, 689–695 (2003).

15. TCR VβRepertoire usage in T cells : Normal Range

16. TCR Vβ Analysis: A case of ATLL

17. Total V beta usage Indirect Clonal Normal Direct Clonal

18. TCR Vβ Analysis: A case of PTCL

19. What levels of expanded T-cell subset as a monoclonal T-cell population? • Suggestions in literature : Expression of a single TCR-Vβ • Lima M et al. Am J Pathol. 2001;159:1861-1868 • > 40% sensitivity 93% & specificity 80% • > 60% sensitivity 81% & specificity 100% • Beck RC et al. Am J ClinPathol. 2003;120:785-794. • 1.6 times of UNL • Morice WG et al. Am J ClinPathol. 2004;121:373-383. • 10-fold or > 50% of the T cells • 40% to 49% suggestive • > 70% failed to react with panel of Ab • B Feng,et al. J ClinPathol 2010 63: 141-146. • > 50% of a gated T cell subset; or • >10 times above its normal ranges T cell subsets; or • >70% of gated T cells failed to react to any of the TCR-Vb antibodies

20. Questions for TCR - V beta analysis 1 – At what level or % – clonal ? 2 – Gating strategy ? 3 – Can we use it in samples like BM, CSF, FNA ? 4 – Can we use it for MRD ?

21. NCI Study – 2007 to 2010 Patients & Samples Concurrent molecular studies for TCRG gene rearrangement by PCR were performed in 36 cases. Tembhare P et al. Am J ClinPathol 2011;135:890-900.

22. Gating Strategy Gating System -1 (GS-1) Gating System -2 (GS-2)

23. Cut off levels for T cells Clonality • We found, • Using aberrant phenotype based gating strategy i.e. GS-2 Demonstration of > 50%usage of single V beta domain OR Failure to demonstrate > 70% of immunoreactivity Tembhare P et al. Am J ClinPathol 2011;135:890-900.

24. TCR-Vβ Monitoring of MRD T NHL • MRD - 61(48 PB, 7 FNA, 4 CSF and 2 BM) • Sequential samples - 14 patients - 3 years • Short immunophenotypic panels and • Single clone specific TCR-Vβ antibody cocktails • Number of tumor cells per micro liter of blood Tembhare P et al. Am J ClinPathol 2011;135:890-900.

25. Follow up

26. Number of cells at each interval

27. The minimum level of clonal T cells - 0.8 cells/ul Ranged from 0.8 cells/ul - 306,603 cells/ul Median value of 1132 cell/ul MRD by TCR-Vβ repertoire

28. TCR-Vβ Monitoring in Paucicellular specimens FNAs and CSF: • Patients: 6 ATLL & 2 PTCL-NOS Tembhare P et al. Am J ClinPathol2012;137:220-226

29. Summary Sensitive method for detection of T cell clonality • Gating strategy – • aberrant immunophenotypes based – superior than T cell subset based. Clonality - > 50% TCR-Vβ subset restriction > 70% negative for all Vβ subsets TCR-Vβ Repertoire kit Useful in low cellularity specimens such as FNA and CSF Quantitative and highly sensitive MRD evaluation Highly sensitive - treatment response & early relapse